Apparatus and method for spotting a substrate

ABSTRACT

The present invention provides a method and apparatus for dispensing a small volume of a selected liquid, such as a biological sample or reagent, onto a substrate. The device includes a tube adapted to contain the liquid. An elongate fiber is disposed within the tube for axial movement therein between raised and lowered positions. Upon shifting or oscillating the fiber between its raised and lowered positions, a liquid spot can be formed at a selected position on the substrate. The device is readily adaptable for the production of micro-arrays having a great number of individual spots.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of application Ser. No.09/812,643, filed Mar. 20, 2001, which is a divisional of applicationSer. No. 09/270,218, filed Mar. 15, 1999, each of which is incorporatedherein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to the dispensing of liquids. Moreparticularly, the invention provides an apparatus and method of use forspotting liquids, such as biological samples or reagents, onto asubstrate.

BACKGROUND OF THE INVENTION

[0003] Target compounds, or analytes, present within a sample can oftenbe identified through the controlled exposure of the sample to anappropriate probe, with subsequent detection of a resulting reaction. Ina typical arrangement, a sample of a test solution containing an analyteof interest is exposed to a probe carrying a detectable reporter. Theprobe is chosen such that it can specifically is bind the analyte, e.g.,by hybridization of complementary nucleotide sequences, orantibody-antigen interactions. After excess probe material has beenremoved, e.g., washed away, specific binding of the probe to the analytecan be detected.

[0004] As the sensitivity of analytical techniques continues to improve,it is increasingly desirable to carry out such analyses using very smallvolumes of samples/reagents. This is especially true in situationsinvolving expensive compounds. Accordingly, it is now popular to utilizevery small volumes of such liquids laid down as “spots” on the surfaceof a substrate, such as a slide, micro-card, or chip.

[0005] Not only is it often desirable to provide ultra-small volumes ofindividual samples and/or reagents in the form of spots, it is becomingincreasingly popular to arrange numerous such spots in close proximityto one another as an array on a substrate. For example, a lab technicianmight need to evaluate a specimen for the presence of a wide assortmentof target biological and/or chemical compounds, or to determine thereaction of many different specimens against one or more reagents, suchas labeled probes. High-density array formats permit many reactions tobe carried out in a substantially simultaneous fashion, saving space,time and money.

[0006] Both manual and automated devices for dispensing very small fluidvolumes have been devised, including, for example, micropipettes, pins,quills and ink-jetting devices. While suitable for some purposes, eachof these is associated with certain disadvantages. For example,micropipettes are generally incapable of accurately dispensing theextremely small volumes of liquid called for by many present-dayprotocols. With regard to pens and quills, a number of problems need tobe resolved relating to the differences in size and shape of the spotswhich are placed (which can lead to differences in resulting signalintensity or overlap of spots), “missed spots” (where little or nosample is placed on the surface), and the overhead associated withcleaning and reloading. Ink-jet devices dispense a controlled volume ofliquid onto a substrate by use of a pressure wave created within thecartridge. This approach is not acceptable for the spotting of samplescontaining relatively fragile macromolecules, as they can become sheeredor otherwise damaged. Further, ink-jetting devices are associated with ahigh degree of splattering, thereby presenting a substantial risk ofcontamination, particularly for closely spaced spots.

[0007] As an additional disadvantage, most of the known spotting devicesrequire very precise placement of the spotting head relative to thesubstrate surface. Variations in the distance between the spotting headand the substrate surface can result in inconsistent spot sizes and/ormissed spots. With particular regard to contact-type devices, if placedto close to the substrate, the spotting tip can collide with thesubstrate surface with a force sufficient to damage the spotting tipand/or the substrate.

[0008] In view of the above, the need is apparent for a device andmethod useful for delivering a micro-volume of liquid onto a substratein a quick and precise manner. Preferably, the device should berelatively easy to use, cost effective and readily adaptable for theproduction of micro-arrays having a great number of individual spots.

SUMMARY OF THE INVENTION

[0009] In one of its aspects, the present invention provides anapparatus for micro-spotting a predetermined volume of a liquid at aplurality of spaced regions on a substrate or substrates.

[0010] In one embodiment, the apparatus includes a tube adapted tocontain a selected liquid, such as a biological sample, reagent, or thelike. The lower end of the tube defines an orifice having a diameter ofless than about 1 mm. In an exemplary construction, the diameter of theorifice is less than about 500 μm, and preferably less than about 200μm. An elongate fiber is disposed within the tube for axial movementtherein between raised and lowered positions. The fiber, which has afree distal end, is provided with a diameter that is less than the innerdiameter of the tube's lower end. In one exemplary arrangement, thediameter of the fiber is between about 10-100 μm smaller than that ofthe orifice. For example, a fiber having a diameter of about 100 μm canbe disposed within a tube having an inner diameter of between about 110to 200 μm. A workpiece holder can be employed to hold a selectedsubstrate or substrates for spotting. At its raised position, thefiber's free end is spaced from the surface of such a substrate. At itslowered position, the fiber's free end contacts the surface of thesubstrate.

[0011] Shifting means are operatively connected to the fiber forshifting the same between its raised and lowered positions. The shiftingmeans can be, for example, an actuator, such as a linear or verticalactuator, or the like. Positioning means are provided for positioningthe tube and associated fiber laterally with respect to the workpieceholder, at selected deposition positions with respect to the substrate.The positioning means can be adapted to move the substrate and/or thetube, fiber and shifting means. In one embodiment, for example, thepositioning means is an x-y positioner (e.g., a robotically controlledx-y movable arm) operatively connected to the tube and the shiftingmeans.

[0012] A control unit is operatively connected to the positioning meansand shifting means for use in successively (i) positioning the tube andassociated fiber at a selected deposition position with respect to asubstrate, and (i) shifting the fiber to its lowered position, todeposit a selected volume of liquid upon such substrate. In a preferredembodiment, the fiber is (i) laterally flexible and (ii) substantiallyincompressible along its longitudinal axis. Suitable fibers having suchcharacteristics include, for example, optical fibers. Advantageously,these characteristics permit efficient transfer of motion from theshifting means to the fiber, and the accommodation of variations in thedistance between the tube's lower end and the substrate by flexing, orbowing, of the fiber.

[0013] According,to one embodiment, the apparatus is adapted for use inmicrospotting a predetermined volume of a liquid at a preselectedposition on each of a plurality of different substrates in the workpieceholder. The control unit, in this embodiment, is operable to positionthe tube successively at such preselected position on each substrate.

[0014] In one embodiment, the tube has a substantially uniform diameter,and includes a larger-diameter upper reservoir for holding the selectedliquid. The tube and reservoir can be separately formed and subsequentlyattached together, or they can be integrally formed.

[0015] In another embodiment, the tube's inner diameter tapers onprogressing downwardly to a defined-volume tube end region having thediameter of the orifice. The diameter of the tube end region, in thisembodiment, is substantially the same as that of the fiber. The fiber'send, with such in its raised position, is disposed above the tube endregion, such that shifting of the fiber from its raised to its loweredposition is effective to expel from the tube the volume of liquidcontained in the tube end region.

[0016] One embodiment of the apparatus, particularly useful inmicro-spotting a predetermined volume of one or more selected liquidssimultaneously at selected deposition regions on a substrate, includes aplurality of tubes, and associated fibers, and shifting means. The tubescan take the form, for example, of channels provided in a manifold.Fiber flexing accommodates variations in the distance between the tubes'lower ends and the associated positions at the substrate.

[0017] The present invention further provides an apparatus formicro-spotting a predetermined volume of a selected liquid on asubstrate, including a tube having an inner diameter that tapers onprogressing downwardly to a defined-volume tube end region having asubstantially uniform diameter of less than about 1 mm. According to oneembodiment, the diameter along the tube end region is less than about500 μm, and preferably less than about 200 μm. The tube is adapted tocontain the selected liquid by capillary or surface tension forces. Anelongate fiber having a diameter substantially the same as that of thetube end region is disposed within the tube for axial movement thereinbetween raised and lowered positions at which the fiber's free end isdisposed above and below the tube end region, respectively. Shiftingmeans (e.g., an actuator, such as a linear or vertical actuator, or thelike) are operatively connected to the fiber for shifting the samebetween its raised and lowered positions, whereby a defined volume ofliquid contained in the tube end region is expelled from the tube onto aselected substrate disposed below the tube.

[0018] In a preferred embodiment, the fiber is (i) laterally flexibleand (ii) substantially incompressible along its longitudinal axis. Forexample, the fiber can be an optical fiber. Advantageously, thesecharacteristics permit efficient transfer of motion from the shiftingmeans to the fiber, and the accommodation of variations in the distancebetween the tube's lower end and the substrate by flexing, or bowing, ofthe fiber.

[0019] According to one embodiment, the fiber makes contact with thesubstrate when shifted to its lowered position. In another embodiment,the fiber remains spaced apart from the substrate when shifted to itslowered position.

[0020] The micro-spotting apparatus can be used to micro-spot apredetermined volume of a liquid at a preselected position at each of aplurality of substrates. In one such embodiment, the apparatus furtherincludes positioning means for positioning the tube and associated fibersuccessively at the preselected position. Fiber flexing accommodatesvariations in the distance between the tube's lower end and thedifferent substrate positions.

[0021] In another embodiment, the apparatus is adapted for use inmicro-spotting a predetermined volume of one or more selected liquidssimultaneously at multiple selected deposition regions on a substrate.In this embodiment, the apparatus further includes a plurality of tubes,and associated fibers and shifting means. In an exemplary arrangement,the tube end regions have diameters of less than about 200 μm, thefibers are flexible fibers, and the fibers in their lowered positionsare adapted to make contact with the substrate. Additionally, fiberflexing accommodates variations in the distance between the tubes' lowerends and the associated positions on the substrate.

[0022] Another aspect of the present invention provides an apparatus forproducing an array of liquid-reagent spots on a substrate.

[0023] In one embodiment, the array-producing apparatus includes amanifold, or channel assembly, having a plurality of capillary channels,each adapted to hold a selected liquid. The channels have oppositeupper-end and lower-end openings, and inner diameters that decrease onprogressing from the upper- to the lower-end openings. The lower-endopenings, in this embodiment, define the pattern and center-to-centerspacing, or pitch, of the spot array. A support is movable betweenraised and lowered positions with respect to the manifold. A pluralityof fibers are suspended from the support for movement therewith. Eachfiber is adapted to move longitudinally within an associated channel, asthe support is moved between its raised and lowered positions. Movementof the fibers from their raised to lowered positions is effective todeposit a selected volume of liquid from each channel in the manifold.

[0024] One embodiment further provides shifting means operativelyconnected to the support for shifting the same between its raised andlowered positions.

[0025] The spacing between adjacent upper-end openings of the manifoldcan be the same as that between adjacent lower end openings, or it candiffer. In one embodiment, the spacing between adjacent upper-endopenings is substantially greater than that between adjacent lower-endopenings. For example, the spacing between lower-end openings can be onehalf, one third, or one fourth that of the upper-end openings.

[0026] According to one embodiment, the fibers are adapted to contact anunderlying substrate, with the support in its lowered position, andvariations in the length of fiber extending between its associatedchannel end and its point of contact on the substrate is accommodated byfiber flexing.

[0027] In one particular construction, the diameter of the channels attheir lower ends is less than about 200 μm, and between about 10-100 μmlarger than that of an associated fiber.

[0028] In another exemplary construction, each channel has asubstantially uniform diameter extending along a lower end region thatterminates at the channel's lower end. Further, the diameter of eachchannel end region is substantially the same as that of the associatedfiber. The fiber's end, with such in its raised position, is disposedabove the channel end region, such that shifting of the fiber from itsraised to its lowered position is effective to expel from the channelthe volume of liquid contained in the channel end region.

[0029] A further aspect of the present invention provides a valvingapparatus for use in metering a selected amount of liquid onto thesurface of a substrate.

[0030] According to one embodiment, the valving apparatus of theinvention includes a reservoir for holding a selected liquid. A tubeextends from the reservoir and terminates at a lower end orificeadjacent a plane adapted to be occupied by the surface of a selectedsubstrate. A fiber is disposed in the tube for axial oscillatorymovement therein, with a lower portion of the fiber extending throughthe orifice. The inner diameter of the tube and the diameter of thefiber are dimensioned to prevent fluid flow through the orifice in theabsence of fiber oscillation. Oscillating means (e.g., an oscillatingunit) operatively connect to the fiber for oscillating the same,including a control unit for determining the oscillation amplitude,frequency and time applied to the fiber, and thereby the amount ofliquid allowed to pass through the tube orifice.

[0031] One embodiment of the valving apparatus further includespositioning means for positioning the tube and fiber with respect to thesubstrate, from one selected lateral position to another. In anexemplary arrangement, the positioning means is operatively connected tothe tube, fiber and oscillating means.

[0032] In one embodiment, the oscillation means is adapted to produce anoscillation frequency of at least about 10 Hertz, and preferably atleast about 100 Hertz. In another embodiment, the oscillation means isadapted to produce an oscillation amplitude of at least about 10 μm, andpreferably at least about 100 μm.

[0033] In one exemplary construction, the tube of the valving apparatushas a lower-end diameter of less than about 100 μm, and the clearancebetween the is fiber and tube at its lower end is less than about 25 μm.

[0034] The fiber of the valving apparatus can remain spaced apart fromthe selected substrate during its oscillation cycle, or it can beadapted to contact the substrate during at least a portion of itsoscillation cycle. In one embodiment, for example, the fiber remains incontact with the substrate throughout its oscillation cycle.

[0035] In another of its aspects, the present invention provides amethod of forming a reagent spot on a substrate. According to oneembodiment, the method includes the steps of: (i) reciprocally moving anelongate, flexible fiber longitudinally within a capillary tube holdinga selected liquid at a frequency and amplitude sufficient to pump aportion of the liquid out through an orifice at a lower end of the tube,thereby forming a pendent drop; and (ii) placing the pendent drop at aselected region on the substrate.

[0036] According to one general embodiment, the pendent drop is placedon the substrate by contacting the drop and/or the tip of the fiber withthe selected region of the substrate. In another embodiment, the pendentdrop is placed on the substrate by maintaining the fiber in spacedrelation over the selected region and enlarging the pendent drop untilit falls under the force of gravity.

[0037] These and other features and advantages of the present inventionwill become clear from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The structure and manner of operation of the invention, togetherwith the further objects and advantages thereof, may best be understoodby reference to the following description taken in conjunction with theaccompanying drawings, in which:

[0039]FIG. 1 is partially schematic, side-elevational view, withportions shown in section, of a spotting device constructed inaccordance with an embodiment of the present invention;

[0040]FIG. 2 is a partially schematic, top plan view showing componentsof an automated apparatus for forming arrays in accordance with theinvention;

[0041]FIGS. 3A to 3C illustrate a spotting device, and method of use,for delivering a micro-volume of liquid onto the surface of a substrate,in accordance with one embodiment of the present invention;

[0042]FIGS. 4A to 4C illustrate a spotting device, and method of use,for delivering a micro-volume of liquid onto the surface of a substrate,in accordance with a further embodiment of the present invention;

[0043]FIG. 5 illustrates a non-contact delivery method for transferringa microvolume of liquid from a tube to the surface of a substrate usinga valving apparatus as taught by the present invention;

[0044]FIG. 6 illustrates a contact delivery method for transferring amicrovolume of liquid from a tube to the surface of a substrate using avalving apparatus as taught by the present invention;

[0045]FIGS. 7A to 7E illustrate a spotting device, and method of use,for delivering a micro-volume of liquid onto the surface of a substrate,according to an embodiment of the present invention;

[0046]FIGS. 8 and 9 are partially schematic, side-elevational views of aspotting head for laying down an array of liquid spots on the surface ofa substrate, according to an embodiment of the present invention;

[0047]FIGS. 10A to 10E illustrate an apparatus, and method of use, fordelivering a liquid reagent from a tube into the well of a microplate,as taught by the present invention; and

[0048]FIG. 11 is a partially schematic, side-elevational view of anautomated system for simultaneously delivering one or more liquidreagents into a plurality of wells of a microplate.

DETAILED DESCRIPTION OF THE INVENTION

[0049] The following discussion of the preferred embodiments of thepresent invention is merely exemplary in nature. Accordingly, thisdiscussion is in no way intended to limit the scope of the invention.

[0050] One aspect of the invention provides a device for dispensing asmall volume of a liquid reagent on a substrate. Generally, the deviceincludes a tube adapted to contain the liquid. An elongate fiber isdisposed within the tube for axial movement therein between raised andlowered positions. Upon shifting or oscillating the fiber between itsraised and lowered positions, a liquid spot can be formed at a selectedposition on the substrate.

[0051] In one exemplary arrangement of a spotting device, denotedgenerally as 8 in FIG. 1, a fiber, indicated as 12, extendslongitudinally within a tube, denoted as 14, having an orifice 16 at itslower end. Tube 14 is adapted to contain a liquid reagent, such as 18,for controlled deposition on a substrate, as discussed more fully below.Tube 14 can be formed, for example, from metal, plastic, glass, ceramic,or other material(s) considered suitable by those skilled in the art. Areservoir 20, disposed in fluid communication with tube 14, is adaptedto receive and hold a supply of liquid reagent. Reservoir 20 feedsliquid reagent to tube 14 as the tube's reagent content is depleted. Inan exemplary construction, a hypodermic needle (gauge 30, ¼ in. long, 90deg. blunt end) is employed as the tube, and an associated plastic luerhub is used as the reservoir.

[0052] Normally, capillary or surface tension forces prevent free flowof the liquid reagent out of the tube's lower orifice. In this regard,the tube's lower region can be of capillary size, so that capillaryforces prevent free flow of liquid reagent 18 out of orifice 16. Forexample, orifice 16, and a region of tube 14 extending upwardlytherefrom, can be formed with an inner diameter of less than about 2 mm,and preferably less than about 1 mm. In one particular construction, theinner diameter along the lower region of tube 14 is less than about 200μm. To further encourage the desired capillary action, thecapillary-size region of tube 14 can be provided with an interiorsurface that is hydrophilic, i.e., wettable. For example, the interiorsurface of tube 14 can be formed of a hydrophilic material and/ortreated to exhibit hydrophilic characteristics. In one embodiment, theinterior surface has native, bound or covalently attached chargedgroups. One suitable surface is a glass surface having an absorbed layerof a polycationic polymer, such as poly-I-lysine.

[0053] Tube 14 can be provided with an exterior surface that ishydrophobic, i.e., one that causes aqueous medium deposited on thesurface to bead. For example, the exterior surface of tube 14 can beformed of a hydrophobic material and/or treated to exhibit hydrophobiccharacteristics. This can be useful, for example, to prevent spreadingof a drop, formed at the region of orifice 16, along the outer surfaceof the tube. It can also be useful to maintain a drop of liquid in theform of a globule at the lowermost tip. A variety of known hydrophobicpolymers, such as polystyrene, polypropylene, and/or polyethylene, canbe utilized to obtain the desired hydrophobic properties. In addition,or as an alternative, a variety of lubricants or other conventionalhydrophobic films can be applied to the tube's exterior surface,particularly along the tube's lower region proximate outlet 16.

[0054] With continuing reference to the embodiment of FIG. 1, thediameter of fiber 12 is between about 10 to 500 μm smaller than theinner diameter of tube 14. In this regard, fiber 12 can have a diameterof between about 25 to 1,000 μm. According to one embodiment, fiber 12has a diameter of less than about 200 μm, and preferably less than about100 μm. In one particular arrangement, a fiber having a diameter ofabout 75 μm extends longitudinally within a tube having an innerdiameter of about 100 μm along its lower region, and an outer diameterof about 200 μm. In another arrangement, a fiber having a diameter ofabout 50 μm extends longitudinally within a tube having an innerdiameter of about 75 μm along its lower region, and an outer diameter ofabout 200 μm.

[0055] For reasons that will become apparent, fiber 12 is preferablyconstructed to be laterally flexible and longitudinally incompressible.Materials suitable for forming fiber 12 include those typically employedin the construction of optical fibers, such as glass, plastic, silica,quartz, and the like. Suitable optical fibers are available from anumber of commercial sources. One particularly preferred fiber, having adiameter of about 0.002 inches (i.e., about 50.8 μm), is that suppliedby Edmund Scientific Co. (Barrington,. N.J.) under catalog # F54014.

[0056] As previously indicated, fiber 12 is disposed within tube 14 foraxial movement therein between raised and lowered positions. In thepresent embodiment, when in the raised position, the fiber's free end ortip 12 a is spaced apart from the surface of a selected substrate, suchas slide 22. When shifted to the lowered position, tip 12 a approachesthe surface of substrate 22.

[0057] Shifting means are operatively connected to fiber 12 for shiftingthe same between its raised and lowered positions. The shifting meanscan include, for example, an actuator that is movable between twopositions, such as a linear or vertical actuator, or the like. In theembodiment of FIG. 1, for example, a solenoid assembly 24 is activatableto draw a solenoid piston 26 downwardly, then release the piston, e.g.,under spring bias, to a normal, raised position (shown in dashed lines).Many solenoids are available from commercial sources, and suitablemodels can be readily chosen by those skilled in the art. One particularsolenoid, contemplated for use herein, is available from JamecoElectronics Components (Belmont, Calif.) under part # 145314 (12 VDCContinuous Tubular Push/Pull Solenoid). In one embodiment, the solenoidis operable to shift the fiber up and down over a stroke of about 1 mm.

[0058] Other actuators, useful for shifting the fiber, include, forexample, pneumatic, hydraulic, magnetostrictive, and piezoelectricactuators, as well as motor assemblies (e.g., steppers) operable togenerate a downward motive force followed by reciprocation. Severalparticular assemblies which can be readily adapted for use herein as theshifting means are disclosed, for example, in U.S. Pat. Nos. 3,164,304;3,329,964; 3,334,354; 5,443,791; 5,525,515; 5,551,487; 5,601,980; and5,807,522; each of which is expressly incorporated herein by reference.

[0059] Positioning means can be utilized to move the spotting devicelinearly or in an x-y plane to position the spotting device at aselected deposition position. In this regard, certain embodimentsprovide for movement of the tube while the target substrate is heldstationary. According to other embodiments, the tube remains in a fixedposition, while the substrate is shuttled into position. Still furtherembodiments provide for movement of both the tube and the targetsubstrate, either sequentially or in parallel.

[0060] In one exemplary arrangement of the positioning means, thespotting device is carried on an arm of an x-y positioner. The arm, inthis arrangement, can be moved either linearly or along an x-y plane toposition the spotting device at a selected deposition position. Suchmovement can be accomplished, for example, using a robotic assembly, orthe like. Exemplary robotic devices include, for example, robots withelectronically controlled linked or crossed movable arms, such as aSCARA, gantry and Cartesian robots. It is understood, of course, thatany other robotic mechanism could be used in accordance with the presentinvention so long as it can accomplish substantially the same purposesand secure substantially the same result. In this regard, cylindricalrobots, polar robots, articulated robots, or the like can be utilized.In one embodiment, the positioning means comprises a motorized x-ycarriage or rail assembly. For example, an AUTOMOVE® 402, available fromAsymtek (Carlsbad, Calif.), can be used for x-y positioning and solenoidactivation.

[0061] The arm that supports the tube, in the above arrangement, caninclude a clamp capable of releasably grasping the tube. Thisarrangement permits swapping of the tube with a different one, e.g.,loaded with a different liquid reagent, as desired.

[0062] One particular positioning means, contemplated for use herein,will now be described in detail. The apparatus is shown in planar, andpartially schematic view in FIG. 2. A spotting device 8 in the apparatushas the basic construction described above with respect to FIG. 1, andincludes a tube 14 terminating at a lower orifice. A fiber 12, disposedaxially within the tube 14 of the spotting device 8, is adapted formovement toward and away from the surface of a substrate, to dispense aselected volume of liquid sample or reagent, as described herein. Asolenoid 24, or other shifting means, effects this movement. Solenoid 24is under the control of a control unit 52 whose operation will bedescribed below.

[0063] The spotting device is carried on an arm 54 that is threadedlymounted on a worm screw 58 driven (rotated) in a desired direction by astepper motor 62 also under the control of unit 52. At its left end inthe figure, screw 58 is carried in a sleeve 64 for rotation about thescrew axis. At its other end, the screw is mounted to the drive shaft ofthe stepper motor, which in turn is carried on a sleeve 66. The spottingdevice, worm screw, the two sleeves mounting the worm screw, and thestepper motor used in moving the device in the “x” (horizontal)direction in the figure form what is referred to here collectively as adisplacement assembly 66.

[0064] The displacement assembly is constructed to produce precise,micro-range movement in the direction of the screw, i.e., along an xaxis in the figure. In one mode, the assembly functions to move thespotting device in x-axis increments having a selected distance in therange 5-500 μm. In another mode, the spotting device may be moved inprecise x-axis increments of several microns or more, for positioningthe spotting device at associated positions on adjacent substrates, aswill be described below.

[0065] The displacement assembly, in turn, is mounted for movement inthe “y” (vertical) axis of the figure, for positioning the spottingdevice at a selected y axis position. The structure mounting theassembly includes a fixed rod 68 mounted rigidly between a pair of framebars 70, 72, and a worm screw 74 mounted for rotation between a pair offrame bars 76, 78. The worm screw is driven (rotated) by a stepper motor80 that operates under the control of unit 52. The motor is mounted onbar 76, as shown.

[0066] The structure just described, including worm screw 74 and motor80, is constructed to produce precise, micro-range movement in thedirection of the screw, i.e., along a y axis in the figure. As above,the structure functions in one mode to move the spotting head in y-axisincrements having a selected distance in the range 5-500 μm, and in asecond mode, to move the spotting head in precise y-axis increments ofseveral microns or more, for positioning the spotting head at associatedpositions on adjacent substrates.

[0067] A workpiece holder 82 in the apparatus functions to hold aplurality of substrates, such as substrates 22 on which the microarraysof reagent regions are to be formed by the apparatus. The holderprovides a number of recessed slots, such as slot 86, which receive thesubstrates, and position them at precise selected positions with respectto the frame bars on which the spotting device positioning means ismounted.

[0068] As noted above, the control unit in the device functions toactuate the two stepper motors and solenoid in a sequence designed forautomated operation of the apparatus in forming a selected microarray ofreagent regions on each of a plurality of substrates.

[0069] The control unit is constructed, according to conventionalmicroprocessor control principles, to provide appropriate signals to thesolenoid and each of the stepper motors, in a given timed sequence andfor an appropriate signaling time. The construction of the unit, and thesettings that are selected by the user to achieve a desired arraypattern, will be understood from the following description of a typicalapparatus operation.

[0070] Initially, one or more substrates are placed in one or more slotsin the holder. Motors 62, 80 are then actuated to position the spottingdevice at a selected array position at the first of the substrates.Solenoid actuation of the spotting device is then effected to dispense aselected-volume aliquot of that reagent at this location. This operationis effective, for example to dispense a selected volume preferably lessthan about 1 μl (e.g., between about 2 pl and 2 nl) of the liquidreagent.

[0071] The spotting device is now moved to the corresponding position atan adjacent substrate and a similar volume of the liquid reagent isdispensed at this position. The process is repeated until the reagenthas been dispensed at this preselected corresponding position on each ofthe substrates.

[0072] Where it is desired to dispense a single reagent at more than twoarray positions on a substrate, the spotting device may be moved todifferent array positions at each substrate, before moving the spottingdevice to a new substrate, or liquid reagent can be dispensed atindividual positions on each substrate, at one selected position, thenthe cycle repeated for each new array position.

[0073] To dispense the next reagent, the spotting device is exchangedfor another such device containing a different selected reagent. Theprocess of dispensing the reagent at each of the correspondingsecond-array positions is then carried out as above. This process isrepeated until an entire microarray of liquid reagents on each of thesubstrates has been formed.

[0074] Several other x-y positioning assemblies which can be readilyadapted for use herein as the positioning means are disclosed, forexample, in U.S. Pat. Nos. 5,443,791; 5,551,487; and 5,587,522; each ofwhich is expressly incorporated herein by reference.

[0075] As previously mentioned, the positioning means can instead, or inaddition, be adapted to move the substrate to a spotting position. Inthis regard, the substrate can be adapted for manipulation by a roboticassembly, or it can be supported on a conveyor, or an x-y movable stageor platform.

[0076] Any desired substrate(s) can be used with the present invention,including slides, cards, plates, chips, and the like. In one generalembodiment, the substrate surface is relatively hydrophilic, i.e.,wettable. For example, the surface can have native, bound or covalentlyattached charged groups. One such surface is a glass surface having anabsorbed layer of a polycationic polymer, such as poly-I-lysine. In oneembodiment, for example, an aqueous or predominantly aqueous reagentsolution or biological sample is spotted onto a slide having ahydrophilic surface. In another embodiment, the substrate surface has oris formed to have a relatively hydrophobic character, i.e., one thatcauses aqueous medium deposited on the surface to bead. A variety ofknown hydrophobic polymers, such as polystyrene, polypropylene, orpolyethylene have desired hydrophobic properties, as do a variety oflubricant or other hydrophobic films that may be applied to thesubstrate surface.

[0077] In some cases, it is desired to spot out the reagents in a humidenvironment so that the droplets do not dry until the arraying operationis complete.

[0078] Several exemplary devices and methods for spotting a substrateare depicted in FIGS. 3 to 6. Generally, a spotting device of theinvention is positioned over a selected region of a substrate. In atypical operation, this is accomplished by aligning the selected regionof the substrate with the spotting device such that the selected regionintersects a line defined by an extrapolation of the spotting device'scentral longitudinal axis. An elongated fiber adapted for movementaxially within the spotting device is then shifted or oscillated betweenits raised and lowered positions in a manner effective to transfer analiquot of liquid from the tube onto the selected region of thesubstrate. It should be noted that any of the above shifting means andpositioning means can be used in connection with the followingembodiments. Also, any suitable control unit, such as 52 in FIG. 2, canbe employed.

[0079] It should also be noted that, as the fiber reciprocates in acycle from its raised position to its lowered position and back, thefiber's terminal end or tip will travel through a path referred toherein as the “stroke.” Along its forward stroke, the tip travels from araised peak to a lowered peak. Along its back stroke, the tip travelsfrom its lowered peak back to its raised peak.

[0080] In one embodiment, shown in FIGS. 3A to 3C, at the beginning ofits stroke, i.e., at its raised peak, the tip 12 a of fiber 12 issituated outside of tube 14, such that a section of fiber 12 intersectsa plane defined by the terminal rim of tube 14 at orifice 16. From thisbeginning position, illustrated in FIG. 3A, the fiber's tip 12 a ismoved toward the surface of a selected substrate, such as slide 22,eventually reaching its lowered peak whereat tip 12 a contacts aselected substrate region 22 a, as shown in FIG. 3B. It should be notedthat fiber 12 carries with it a layer of liquid reagent 18 on itsexterior surface, including at the surface of tip 12 a. Upon contactingthe substrate, a small and controlled portion of liquid reagent isdelivered from tip 12 a to the selected region 22 a of substrate 22.After contacting substrate 22, fiber 12 is shifted back to its raisedposition, leaving behind a spot of liquid, as at 28, on the substrate'ssurface. The spotting device can then be positioned over anotherselected region to lay down an lo additional spot, if desired. Therepetition rate employed can be a few strokes per second, e.g., withinthe range of about 1-10 Hz. In one embodiment, the repetition rate isabout 5 strokes per second.

[0081] It should be appreciated that an exacting tolerance between thespotting device and substrate is not critical to achieve successfulresults using the spotting device of the present invention. That is, thefiber can retreat back to its raised position after only barely touchingthe substrate surface, or the fiber can be lowered farther than what isnecessary toward the substrate surface in order to bring its tip intocontact therewith. Advantageously, the flexibility of the fiber permitsthe fiber to flex, or bow, once the tip abuts the substrate surface.That is, flexing of the fiber can accommodate variations in the distancebetween the lower end of the spotting device and the surface of thesubstrate. This is shown in exaggerated fashion in FIG. 3B forconvenience of illustration.

[0082] It should also be appreciated that the longitudinalincompressibility of the fiber provides for the efficient transmissionof motion from the shifting means to the fiber's tip. Advantageously,this property permits the use of fibers of varying lengths, includingrelatively long fibers (e.g., 10, 20, 30 cm, or more).

[0083] In another embodiment (not shown), similar to the embodiment justdescribed, the forward stroke can bring the fiber's tip very close tothe selected surface region of the substrate, without actually makingcontact. This permits liquid reagent on the fiber's terminal end totouch and adhere to the substrate surface, while actual physical contactbetween the tip itself and the substrate surface is avoided. Whileadvantageous for certain purposes, it will be appreciated that thisembodiment will generally be less desirable than the previouslydescribed embodiment, since an exacting tolerance between the spottingdevice and the substrate surface is required in this case.

[0084] Another spotting method is depicted in FIGS. 4A to 4C. Here, atthe beginning of its stroke, i.e., at its raised peak, the tip 12 a offiber 12 is situated within tube 14, above the plane defined by theterminal rim of tube 14 at orifice 16. From this beginning position,illustrated in FIG. 4A, the fiber tip 12 a is moved toward the surfaceof a selected substrate, such as slide 22. Plunger- or piston-likeaction of tip 12 a acts to push an aliquot of liquid reagent 18 from thelower end region of tube 14. The amount of liquid pushed out in thismanner will depend, in part, on the volume of liquid occupying theregion under tip 12 a at the time tip 12 a is moved toward substrate 22.Thus, the location of the tip's raised peak within the tube, in thisembodiment, will typically be determined, at least in part, by thequantity of liquid that one desires to spot on a substrate. Othervariables that can be adjusted in order to control the amount of liquiddeposited in this embodiment include the surface area of tip 12 a, andthe distance of the gap separating the fiber from the tube's interiorsurface.

[0085] As tip 12 a is moved toward substrate through its stroke, iteventually reaches its lowered peak, in contact with substrate 22. Aftercontacting substrate 22, fiber 12 is shifted back to its raisedposition, leaving behind a spot of liquid, as at 28 in FIG. 4C, on thesubstrate's surface. The spotting device can then be positioned overanother selected region to lay down an additional spot, if desired. Aswith the previously described embodiment, the repetition rate employedcan be a few strokes per second, e.g., within the range of about 1-10Hz. In one embodiment, the repetition rate is about 5 strokes persecond.

[0086] It should be appreciated that the piston-like action of fiber 12,as just described, causes a thicker layer of liquid to form at thefiber's lower end region, as compared to the embodiment of FIGS. 3A to3C. Thus, it is contemplated that this embodiment will be used to createreagent spots having a greater volume.

[0087] In another embodiment (not shown), similar to the embodiment justdescribed, the fiber's tip can be moved toward the selected surfaceregion of the substrate, without making contact. For example, movementof the fiber tip toward the substrate can be abruptly stopped, orreversed, so that liquid is thrown or ejected, e.g., as by inertia, fromthe fiber onto the substrate. Or, the fiber tip can be moved into veryclose proximity to the substrate surface, without physically contactingit, so that liquid reagent carried on the fiber touches and adheres tothe substrate.

[0088] A further aspect of the present invention provides a valvingapparatus for use in metering a selected amount of liquid onto thesurface of a substrate. In one embodiment, shown in FIGS. 5 and 6, atube 14 communicates at its upper end with a reagent-supply reservoir20, and terminates at a lower end orifice 16, much like the previouslydescribed spotting devices. A fiber 12 is disposed in tube 14 for axialoscillatory movement therein.

[0089] The inner diameter of tube 14 and the diameter of fiber 12 aredimensioned to prevent fluid flow through orifice 16 in the absence offiber oscillation. In one embodiment, for example, tube 14 has alower-end diameter of less than about 200 μm, and preferably less thanabout 100 μm, and the clearance between the fiber and tube at its lowerend is less than about 50 μm, and preferably less than about 25 μm.

[0090] Oscillating means operatively connect to fiber 12 for oscillatingit. The oscillating means can comprise, or example, an oscillating unithaving a control unit for determining the oscillation amplitude,frequency and time applied to the fiber, and thereby the amount ofliquid allowed to pass through orifice 16. The oscillating unit cancomprise any device capable of oscillating the fiber axially within thetube in such a controlled manner. Suitable oscillating units caninclude, for example, a solenoid or motor assembly, or a pneumatic,hydraulic, magnetostrictive, or piezoelectric actuator. In oneembodiment, the oscillating means is adapted to produce an oscillationfrequency of at least about 10 Hz, and preferably at least about 100 Hz.Preferably, the oscillating means is adapted to produce an oscillationamplitude of at least about 10 μm, and preferably at least about 100 μm.

[0091] The valving apparatus can further include positioning means forpositioning the tube and fiber with respect to the substrate, from oneselected lateral position to another. In one embodiment, the positioningmeans is operatively connected to the tube, fiber and oscillating means.The positioning means can comprise devices as set forth above withregard to the spotting device.

[0092] In operation, fiber 12 can be moved axially within theliquid-holding tube in a reciprocal fashion. Such oscillatory movementoccurs at a frequency and amplitude, and for a length of time,sufficient to pump a selected quantity of liquid reagent 18 out throughorifice 16 at a lower end of tube 14, thereby forming a pendent drop,such as at 18 a in FIG. 5. The pendent drop can be placed at a selectedregion of a substrate by contacting the drop 18 a with the selectedsubstrate region. In one embodiment, the fiber's tip 12 a is moved intocontact with the substrate. Such contact can be periodic, e.g., once peroscillation of the fiber, or it can be continuous such that the fiberengages the substrate throughout its oscillation cycle. It should beappreciated that formation of such a “liquid bridge,” as illustrated inFIG. 6, permits the creation of relatively large reagent spots.Alternatively, the pendent drop can be placed on the substrate bymaintaining the fiber in spaced relation over the selected region andenlarging the pendent drop until it falls under the force of gravity.

[0093] Another exemplary spotting device of the present invention isdepicted in FIGS. 7A to 7E. Similar to the previous embodiments, aflexible fiber 12 extends longitudinally through a tube 14 having alower orifice 16. In this embodiment, however, the inner diameter oftube 14 tapers on progressing downwardly to a defined-volume tube endregion, indicated generally at 14 a, having a substantially uniformdiameter. Preferably, the tube end region 14 a diameter is substantiallythe same as the diameter at orifice 16. Further, the inner diameteralong the lower end region 14 a of tube 14, proximate orifice 16, isvery close to (e.g., within about 10 μm), and preferably substantiallythe same as, the diameter of fiber 12. For example, in one embodiment,both the fiber's diameter and the inner diameter of the tube, alongregion 14 a, are less than about 200 μm; and are preferably about 100μm. In another embodiment, both of these values are about 50 μm. Theupper, larger diameter portion of the tube can act as a supply reservoir20 for feeding liquid reagent to the lower tube portion as spottingoperations are effected.

[0094] Shifting means are operatively connected to fiber 12 for shiftingthe fiber between its raised and lowered positions. The apparatus canfurther include positioning means for positioning the tube and fiberwith respect to the substrate, from one selected lateral position toanother. In one embodiment, the positioning means operatively connect tothe tube, fiber and oscillating means. The shifting means and thepositioning means can comprise devices as previously set forth herein.

[0095] Steps of a typical operation are depicted sequentially in FIGS.7A through 7E. Initially, the shifting means (not shown) shifts fiber 12from its raised position, of FIG. 7A, towards a substrate, such as slide22. Notably, when fiber 12 is in its raised position, its tip 12 a isdisposed above the tube end region 14 a. As fiber 12 is shifted from itsraised to its lowered position, the volume of liquid reagent 18occupying the tube end region 14 a is expelled from tube 14, as depictedin FIG. 7B. When fiber 12 reaches its lowered position, as shown in FIG.7C, the expelled liquid reagent is transferred to a selected region 22 aof substrate 22. As fiber 12 is shifted back to its raised position, asshown in FIGS. 7D and 7E, a spot of liquid reagent 28 is left behind onthe surface of substrate 22. When the fiber is fully retracted back intothe fluid container (FIG. 7E), another deposition cycle is ready tobegin, if desired.

[0096] As with those previous embodiments involving contact between thefiber and the substrate, it should be appreciated that an exactingtolerance between the spotting device and substrate is not critical toachieve successful results using the just-described spotting device.Advantageously, the flexibility of the fiber permits the fiber to flex,or bow, once the tip abuts the substrate surface, as shown inexaggerated fashion in FIG. 7C for convenience of illustration. That is,flexing of the fiber can accommodate variations in the distance betweenthe lower end of the spotting device and the surface of the substrate.It should also be appreciated that the longitudinal incompressibility ofthe fiber provides for the efficient transmission of motion from theshifting means to the fiber's tip.

[0097] One embodiment provides an operation substantially like that ofFIGS. 7A to 7E, except that the fiber's lower tip never makes contactwith the substrate surface. For example, movement of the fiber tiptoward the substrate can be abruptly stopped, or reversed, so thatliquid is thrown or ejected, e.g., as by inertia, from the fiber ontothe substrate. Or, the fiber tip can be moved into very close proximityto the substrate surface, without physically contacting it, so thatliquid reagent expelled by the fiber touches and adheres to thesubstrate.

[0098] Still a further aspect of the present invention provides ahand-operable spotting device (not shown). The tube, in this embodiment,is dimensioned to fit comfortably in the hand of an operator. In thisregard, the outer dimensions of the hand device are preferably similarto those of typical writing implements, such as ink pens, mechanicalpencils, and the like. For example, the tube can have an outer diameterof between about 0.75-1.50 cm and a length of between about 10-20 cm. Anelongate, flexible fiber, e.g., an optical fiber, is disposed within thetube for axial movement therein between raised and lowered positions.The tube is adapted to hold, e.g., by way of capillary forces, aselected liquid reagent for deposition. The interior dimensions of thetube, and the dimensions of the fiber, can be as set out for any of theforegoing embodiments.

[0099] The fiber of the hand device attaches at its upper end to apiston that is adapted for reciprocal movement within the tube.Normally, the piston is urged away from a lower orifice of the tube,toward the top of the tube, by a coil spring or other biasing mechanism.At this position, the fiber is shifted toward its raised position,substantially retracted into the tube. The shifting means, in thisembodiment, includes a depressible shaft having an end region thatprotrudes through an opening at the upper end of the tube. The other endof the shaft, located within the tube, operatively engages the piston.Upon depressing the rod, e.g., using the thumb of an operator's graspinghand, the normal biasing force of the coil spring can be overcome, sothat the piston is pushed downwardly through the tube. Such movement ofthe piston causes the fiber to move axially within the tube, so that thefiber's lower end protrudes from the tube's lower orifice. At thisposition, a liquid spot can be transferred from the fiber's tip to aselected position on a substrate. Upon releasing the depressible shaft,the coil spring returns the fiber to its raised position.

[0100] In another of its aspects, the present invention provides aspotting head for producing an array of liquid-reagent spots on asubstrate. As discussed more fully below, the spotting head of theinvention can be adapted to lay a great number, e.g., hundreds orthousands, of spots per second.

[0101] One embodiment of the spotting head is shown in FIG. 8. In thisembodiment, a conduit or channel assembly 101, also referred to hereinas a manifold, includes a plurality of channels, such as 114 a-114 b.Each channel 114 a, 114 b has opposite upper-end and lower-end openings,as at 115 a-115 b and 116 a-116 b, respectively. The channels aremaintained in fixed, spaced relation to one another. In one embodiment,the channels take the form of tubes, barrels or funnels that are secured(e.g., snap fit) into a frame or rack body. In another embodiment, thechannels are of a monolithic construction. For example, the channelassembly can be integrally constructed of plastic using an injectionmolding process; or each channel can be formed by boring through a blockof material, such as glass, plastic, metal, or the like.

[0102] The inner diameter of each channel decreases on progressing fromthe upper- to the lower-end openings. For example, the channels can begenerally cone-shaped or horn-shaped channels. The longitudinal axis ofeach channel can be straight, angled, curved, or other suitable shape.In this regard, attention is directed to the generally S-shaped channelsshown in FIG. 8.

[0103] A region of each channel extending from a respective one of thelower-end openings is of capillary size, such that a liquid, e.g., abiological sample or reagent solution, placed in the channel willnormally be maintained therein by way of capillary forces. Any innerdiameter that effects the desired capillary action can be utilizedwithin the scope of this invention. For example, the capillary-sizeregions can be formed with an inner diameter of less than about 1 mm,and preferably less than about 200 μm. To further encourage the desiredcapillary action, the capillary-size region of each conduit can beprovided with an interior surface that is hydrophilic. While only sixchannels, arranged side-by-side in a linear fashion, are visible in theview of FIG. 8, it should be understood that any reasonable number ofchannels can be disposed in any desired spatial configuration. Forexample, the manifold can include 24, 48, 96, 384, 1024, 1536 channels,or more. In such arrangements, the channel upper- and lower-end openingswill typically be arranged in a regular array, e.g., an 8×12, 16×24,32×32, or a 32×48 array, though other layouts are possible.

[0104] A support, denoted as 105, is adapted for movement between raisedand lowered positions with respect to manifold 101. In the embodiment ofFIG. 8, this is accomplished by providing a frame, such as 107, having atrack comprised of spaced-apart, parallel linear rail portions, denotedas 109 a and 109 b, along which support 105 is guided. For example,support 105 can have a groove or slot (not shown) formed along each ofits side-end regions, proximate rails 109 a, 109 b, with each slot beingslidably mounted over a respective one of the rails.

[0105] Movement and positioning of support 105 along the track can beeffected by way of manual or automatic shifting means. In this regard,and with additional reference to FIG. 9, a motor assembly 121communicates with a controller 123 and power supply 125. A flexible wireor line 127 extends between motor 121 and support 105. One end of wire127 is connected to an upper side of support 105. The other end of wire127 is secured to a spool (not shown) which, in turn, is adapted forrotation by motor assembly 121. In one mode, motor assembly 121 canrotate the spool so as to wind wire 127 therearound, thereby movingsupport 105 up along the track towards its raised position (FIG. 8). Inanother mode, motor assembly 121 can rotate the spool so as to unwind,and thus extend, wire 127 therefrom, thereby moving support 105 down thetrack towards its lowered position (FIG. 9). Rather than employing awire to communicate the motor with the support, other embodimentscontemplate the use of gear assemblies. It should be noted that otherautomatic shifting means, suitable for use herein, include, for example,hydraulic or pneumatic actuators. Alternatively, support 105 can beshifted by hand.

[0106] A plurality of fibers, such as optical fibers 112 a-112 b, arecarried on support 105 for movement therewith. The fibers can be securedto the support in any suitable manner. For example, the upper end regionof each fiber can be received within a respective bore extending up fromthe lower side of support 105. Each fiber's upper end can be held in itsbore, for example, by way of frictional forces and/or by usingconventional adhesives. The fibers are arranged such that the spacingbetween adjacent fibers substantially matches the spacing betweenadjacent upper-end openings of manifold 101, allowing insertion of onefiber per channel as support 105 is moved towards its lowered position.In a typical arrangement, the fibers will be disposed in a regulararray.

[0107] The diameter of the fibers, extending from support 105, as wellas the interior dimensions of the capillary-size regions of thechannels, can be like that set out for any of the previous embodiments.In an exemplary arrangement, the diameter of each channel is less thanabout 200 μm, and each channel is between about 10-100 μm larger thanthat of an associated fiber. In another arrangement, the interiordiameter along the lower end region of each channel is substantially thesame as that of an associated fiber.

[0108] Upon moving support 105 to its lowered position, the lower tip ofeach fiber is passed through a respective channel of manifold 101 andbrought to a plane adapted to be occupied by a substrate. As shown inFIG. 9, when a substrate, such as plate 22, occupies such plane, thefiber tips abut the substrate's surface at this position. In this way,each fiber can transfer an aliquot of a liquid reagent held in itsrespective channel to the surface of the substrate. It should be notedthat the lower-end opening array of manifold 101 defines the array ofspots formed on substrate 22.

[0109] As best viewed in FIG. 8, the fibers extending from support 105are not all of the same length. Rather, they are cut such that uponbeing lowered through their respective channels they will contact anunderlying substrate at roughly the same time, or within a short time ofone another. Appropriate fiber lengths can be established by passing thefibers through the channels, and then cutting each fiber at its regionintersecting the plane adapted to be occupied by the surface of asubstrate. It is not critical to the successful operation of theinvention that the various fibers be cut with exacting precisionrespective to the substrate plane, since variations in the length offiber extending between its associated channel end and its point ofcontact on the substrate can be accommodated by fiber flexing.

[0110] For applications requiring formation of an array of reagent spotshaving the same center-to-center spacing, or pitch, between adjacentspots as between adjacent fibers of the fiber array, the pitch of boththe upper- and lower-opening arrays can be made about equal. Forexample, in one embodiment, each of (i) the fiber array, (ii) theupper-end opening array, and (iii) the lower-end opening array all havesubstantially the same center-to-center spacing. For applicationsrequiring the formation of a reagent spot array having a reduced pitchas compared to the fiber array, on the other hand, it will beadvantageous to utilize a channel assembly having upper- and lower-endopening arrays that differ in pitch, as well. In one exemplaryarrangement of this type, the channel lower-end opening array isprovided with a center-to-center pitch that is smaller than that of theupper-end opening array. FIGS. 8 and 9 show, for example, an embodimentwherein the pitch of the lower-end opening array is substantiallysmaller than that of the upper-end opening array. For example, thecenter-to-center pitch of the lower-end opening array can be betweenabout ½ to ¼ that of the upper-end opening array. In one particularembodiment, the center-to-center spacing of the lower-end opening arrayis about ⅓ that of the upper-end opening array.

[0111] According to one embodiment, a plurality of substrates aresequentially shuttled under the spotting head. For example, a conveyorcan carry a number of tandemly-arranged substrates along a transportpathway passing under a manifold. The channels of the manifold can holda single type of liquid reagent, or multiple types of liquid reagents.Upon positioning a selected substrate beneath the manifold, the conveyorcan pause. At this point, the fibers can be shifted between their raisedand lowered positions to lay down an array of spots on the substrate. Ifdesired, such shifting can be repeated one or more times to transferadditional liquid to the substrate. Such additional liquid can be placedat the already-laid spots, or, upon incrementally moving the substratelaterally under the manifold, at previously unspotted regions of thesubstrate. The just-spotted substrate can then be moved out from underthe manifold and a new, upstream substrate can be moved into positionfor spotting. If desired, several spotting heads can be situated atrespective positions along the transport pathway. In one embodiment, aconveyor shuttles one or more substrates along a transport pathwayextending under several spotting heads that are disposed sequentiallyalong the transport pathway, at positions that are laterally offset fromone another. This arrangement can provide a very compact interleaving ofspots on a given substrate, even though the spots laid by any onespotting head have a wider spacing.

[0112] Changeover from one set of liquid reagents to a different set, orreplacement of an emptied manifold with a loaded one, can beaccomplished in a quick and efficient manner. For example, an operatoror robot can simply remove the present manifold and insert another inits place. In this regard, the manifold can be configured to removablysnap-lock into the frame. Where one manifold holding a first set ofliquid reagents is swapped for another manifold holding a second,different set of reagents, the fiber array can be readily changed aswell. To this end, the fiber support can also be constructed toremovably snap-fit into the frame.

[0113] Refilling low or emptied channels of the manifold can also bereadily accomplished. In this regard, the upper-end openings of thechannels can be dimensioned large enough (e.g., >3 mm diameter) topermit ready access to conventional means of fluid loading, such aspipettes or syringes.

[0114] In one embodiment, both the manifold and the fiber support areconstructed of relatively inexpensive materials, e.g., plastics, metalor glass, using conventional tooling and/or molding procedures. Bykeeping the component cost low, it can be cost-effective to throw awaythe fiber support (with the fibers) and/or the manifold when a newliquid reagent set is introduced. Disposing of one or both of thesecomponents, rather than cleaning and re-using them, eliminates apotential source of contamination. Furthermore, utilization ofdisposable components helps avoid the time, equipment, and labor costsassociated with cleaning/drying efforts. In contrast, most conventionalspotting systems, such as quill, ink-jet, or pin, must be cleaned eachtime a new fluid is deposited.

[0115] The above-described spotting devices and methods provide arelatively low-energy approach to liquid deposition. For example, thespotting head can employ a highly parallel approach to lay many spots(e.g., hundreds or thousands) per second. Notably, the process of layingany single spot is a relatively slow process. For example, eachindividual fiber might lay only one or a few spots per second.Consequently, problems associated with very energetic spot depositionsuch as splattering and misdirected ejection (satellites), andcontamination resulting therefrom, are avoided by the present invention.

[0116] The spotting devices described herein offer reduced reagent lossas compared to most conventional deposition systems. According to thepresent invention, liquids that are deposited onto a substrate aredirectly transferred from a tube or channel onto the surface of asubstrate without the use intermediate containers. It should beappreciated that intermediate containers typically waste fluid becauseof residues and films that are unavoidably left behind. For applicationsrequiring very small amounts of fluid (e.g., a micro-liter or less),intermediate containers such as reservoirs in ink-jets or the split in aquill can waste an unacceptable amount of fluid.

[0117] Still a further aspect of the present invention provides a methodand apparatus for dispensing a liquid reagent into a well or depressionformed, for example, in a tray or plate. With reference to FIGS. 10A to10E, a protrusion, such as a spike 217, extends upwardly from the bottomof a well 219 of a multi-well tray 222. A liquid container, such as anelongate tube 216, holds a liquid 218 for dispensing. Tube 216 isadapted to hold the liquid by capillary or surface tension forces. Asshown in FIGS. 10A to 10B, a meniscus 218 a can form at the lower regionof tube 216. Liquid 218 is dispensed by shifting tube 216 toward well219 until spike 217 pierces meniscus 218 a. The spike, which preferablyhas a wettable surface, draws liquid from the tube into the well.

[0118]FIG. 11 shows an automated system for simultaneously deliveringone or more liquid reagents into a plurality of wells of a plate ortray. The system includes a manifold or channel assembly 301, similar tothat described above with respect to FIGS. 8 and 9, adapted for shiftingbetween raised and lowered positions over a substrate, such asmulti-well tray 322. Manifold 301 includes a plurality of channels, suchas 314 a and 314 b, each having a lower-end region substantially likethe interior of tube 216 of FIGS. 10A to 10E. Shifting means areoperable to shift manifold 301 between its raised and lowered positions.In the embodiment of FIG. 11, for example, a support 305 is configuredto releasably engage an upper region of manifold 301. Support 305, inturn, is adapted to ride along a pair of parallel rails 309 a, 309 bprovided on a frame assembly 307. A motor 321, controller 323, and powersupply 325 are operable to move support 305, and thus manifold 301, upand down along rails 309 a, 309 b via wire 327. Although not visible inFIG. 11, each well of tray 322 includes a protrusion, such as a spike,extending upwardly from its floor. The spikes are adapted to extractliquid reagent from respective channels when manifold 301 is shiftedtowards its lowered position in a fashion substantially as shown inFIGS. 10A-10E.

[0119] Those skilled in the art can now appreciate from the foregoingdescription that the broad teachings of the present invention can beimplemented in a variety of forms. Therefore, while this invention hasbeen described in connection with particular embodiments and examplesthereof, the true scope of the invention should not be so limited.Various changes and modification may be made without departing from thescope of the invention, as defined by the appended claims.

It is claimed:
 1. An apparatus for micro-spotting a predetermined volumeof a liquid at a plurality of spaced regions on a substrate orsubstrates, comprising: a tube adapted to contain such liquid, andhaving a lower end defining an orifice with an inner diameter less thanabout 200 μm; a workpiece holder for holding such substrate(s), anelongate, flexible fiber having a diameter of less than about 100 μm anda distal free end, said fiber being disposed within said tube for axialmovement therein between raised and lowered positions at which thefiber's free end is spaced from and in contact with the surface of sucha substrate(s), respectively, with the substrate(s) held in theworkpiece holder, shifting means operatively connected to said fiber forshifting the same between its raised and lowered positions, positioningmeans for positioning said tube and associated fiber laterally withrespect to the holder, at selected deposition positions with respect tothe substrate(s), and a control unit operatively connected to thepositioning means and shifting means for use in successively (i)positioning the tube and associated fiber at a selected depositionposition with respect to a substrate, and (i) shifting the fiber to itslowered position, to deposit a selected volume of liquid upon suchsubstrate, with variations in the distance between the tube's lower endand the substrate being accommodated by flexing of the fiber.
 2. Theapparatus of claim 1, wherein said positioning means is operativelyconnected to said tube and said shifting means.
 3. The apparatus ofclaim 1, for use in micro-spotting a predetermined volume of a liquid ata preselected position on each of a plurality of different substrates inthe workpiece holder, wherein the control unit is operable to positionthe tube successively at such preselected position on each substrate. 4.The apparatus of claim 1, wherein the diameter of the orifice is betweenabout 10-100 μm larger than that of the fiber.
 5. The apparatus of claim1, wherein the tube has a substantially uniform diameter, and furtherincludes a larger-diameter upper reservoir for holding such liquid. 6.The apparatus of claim 1, wherein the tube's inner diameter tapers onprogressing downwardly to a defined-volume tube end region having thediameter of said orifice, the diameter of the tube end region issubstantially the same as that of the fiber, and the fiber's end, withsuch in its raised position, is disposed above the tube end region, suchthat shifting of the fiber from its raised to its lowered position iseffective to expel from the tube a volume of liquid contained in saidtube end region.
 7. The apparatus of claim 1, for use in micro-spottinga predetermined volume of such a liquid simultaneously at selecteddeposition regions on a substrate, which further includes a plurality ofsuch tubes, and associated fibers and shifting means.
 8. An apparatusfor micro-spotting a predetermined volume of a liquid on a substrate,comprising: a tube adapted to contain such liquid, and having an innerdiameter that tapers on progressing downwardly to a defined-volume tubeend region having a substantially uniform diameter of less than about500 μm, an elongate fiber having a diameter substantially the same asthat of said tube end region, said fiber being disposed within said tubefor axial movement therein between raised and lowered positions at whichthe fiber's free end is disposed above and below said tube end region,respectively, and shifting means operatively connected to said fiber forshifting the same between its raised and lowered positions, whereby adefined volume of liquid contained in said tube end region is expelledfrom the tube onto such a substrate disposed below the tube.
 9. Theapparatus of claim 8, wherein the tube end region has a diameter of lessthan about 200 μm.
 10. The apparatus of claim 9, wherein said fiber is aflexible fiber, and the fiber in its lowered position is adapted to makecontact with such a substrate.
 11. The apparatus of claim 10, for use inmicro-spotting a predetermined volume of a liquid at a preselectedposition at each of a plurality of substrates, wherein said apparatusfurther includes positioning means for positioning said tube andassociated fiber successively at said preselected position, andvariations in the distance between the tube's lower end and thedifferent substrate positions is accommodated by fiber flexing.
 12. Theapparatus of claim 8, for use in micro-spotting a predetermined volumeof such a liquid simultaneously at multiple selected deposition regionson a substrate, which further includes a plurality of such tubes, andassociated fibers and shifting means.
 13. The apparatus of claim 12,wherein the tube end regions have diameters of less than about 200 μm,said fibers are flexible fibers, and the fibers in their loweredpositions are adapted to make contact with such a substrate, withvariations in the distance between the tubes' lower ends and theassociated positions on the substrate being accommodated by fiberflexing.
 14. An apparatus for producing an array of liquid-reagent spotson a substrate, comprising: a manifold having a plurality of capillarychannels, each adapted to hold a selected liquid, said channels havingopposite upper-end and lower-end openings, and inner diameters whichdecrease on progressing from the upper- to the lower-end openings, withsaid lower openings forming said array, a support movable between raisedand lowered positions with respect to said manifold, and a plurality offibers carried on said support for movement therewith, each fiber beingadapted to move longitudinally within an associated channel, as thesupport is moved between its raised and lowered positions, with movementof the fibers from their raised to lowered positions being effective todeposit a selected volume of liquid from each channel in the manifold.15. The apparatus of claim 14, wherein the spacing between adjacentupper-end openings is greater than that between adjacent lower-endopenings.
 16. The apparatus of claim 14, which further includes shiftingmeans operatively connected to the support for shifting the same betweenits raised and lowered positions.
 17. The apparatus of claim 14, whereinthe diameter of the channels at their lower ends is less than about 200μm, and from about 10 to about 100 μm larger than that of the associatedfiber.
 18. The apparatus of claim 17, wherein the fibers are adapted tocontact such a substrate, with the support in its lowered position, andvariations in the length of fiber extending between its associatedchannel end and its point of contact on the substrate is accommodated byfiber flexing.
 19. The apparatus of claim 14, wherein each channel has asubstantially uniform diameter extending along a lower end region thatterminates at the channel's lower end, the diameter of each channel endregion is substantially the same as that of the associated fiber, andthe fiber's end, with such in its raised position, is disposed above thechannel end region, such that shifting of the fiber from its raised toits lowered position is effective to expel from the channel the volumeof liquid contained in said channel end region.
 20. Valving apparatusfor use in metering a selected amount of liquid onto the surface of asubstrate, comprising a reservoir for holding such liquid, a tubeextending from the reservoir and terminating at a lower end orificeadjacent a plane adapted to be occupied by the surface of such asubstrate, a fiber disposed in said tube for axial oscillatory movementtherein, with a lower portion of the fiber extending through theorifice, and the inner diameter of said tube and the diameter of saidfiber being dimensioned to prevent fluid flow through said orifice inthe absence of fiber oscillation, oscillating means operativelyconnected to said fiber for oscillating the same, including a controlunit for determining the oscillation amplitude, frequency and timeapplied to said fiber, and thereby the amount of liquid allowed to passthrough the tube orifice.
 21. The apparatus of claim 20, wherein saidoscillation means is adapted to produce an oscillation frequency of atleast about 10 Hertz.
 22. The apparatus of claim 21, wherein saidoscillation means is adapted to produce an oscillation frequency of atleast about 100 Hertz.
 23. The apparatus of claim 20, wherein saidoscillation means is adapted to produce an oscillation amplitude of atleast about 10 μm.
 24. The apparatus of claim 23, wherein saidoscillation means is adapted to produce an oscillation amplitude of atleast about 100 μm.
 25. The apparatus of claim 20, wherein said tube hasa lower-end diameter of less than about 100 μm, and the clearancebetween the fiber and tube at its lower end is less than about 25 μm.26. The apparatus of claim 20, wherein said fiber is adapted to contactsuch a substrate during at least a portion of its oscillation cycle. 27.The apparatus of claim 26, wherein said fiber is adapted to remain incontact with said substrate throughout its oscillation cycle.
 28. Theapparatus of claim 20, which further includes positioning means forpositioning said tube and fiber with respect to the substrate, from oneselected lateral position to another.
 29. The apparatus of claim 28,wherein said positioning means is operatively connected to said tube,fiber and oscillating means.
 30. A method of forming a reagent spot on asubstrate, comprising: reciprocally moving an elongate, flexible fiberlongitudinally within a capillary tube holding a selected liquid at afrequency and amplitude sufficient to pump a portion of the liquid outthrough an orifice at a lower end of the tube, thereby forming a pendentdrop; and placing the pendent drop at a selected region on thesubstrate.
 31. The method of claim 30, wherein the pendent drop isplaced on the substrate by contacting an end of the fiber with theselected region of the substrate.
 32. The method of claim 30, whereinthe pendent drop is placed on the substrate by maintaining the fiber inspaced relation over the selected region and enlarging the pendent dropuntil it falls under the force of gravity.
 33. An apparatus formicro-spotting a predetermined volume of a liquid at a plurality ofspaced regions on a substrate or substrates, comprising: a tube adaptedto contain such liquid, and having a lower end defining an orifice withan inner diameter less than about 200 μm; a workpiece holder for holdingsuch substrate(s), an elongate, flexible fiber having a diameter of lessthan about 100 μm and a distal free end, said fiber being disposedwithin said tube for axial movement therein between raised and loweredpositions at which the fiber's free end is spaced from and in contactwith the surface of such a substrate(s), respectively, with thesubstrate(s) held in the workpiece holder, an actuator movable betweentwo positions, said actuator operatively connected to said fiber forshifting the same between its raised and lowered positions, and an x-ypositioner operatively connected to said tube for positioning said tubeand associated fiber laterally with respect to the holder, at selecteddeposition positions with respect to the substrate(s), and a controlunit operatively connected to the x-y positioner and vertical actual foruse in successively (i) positioning the tube and associated fiber at aselected deposition position with respect to a substrate, and (i)shifting the fiber to its lowered position, to deposit a selected volumeof liquid upon such substrate, with variations in the distance betweenthe tube's lower end and the substrate being accommodated by flexing ofthe fiber.
 34. An apparatus for micro-spotting a predetermined volume ofa liquid on a substrate, comprising: a tube adapted to contain suchliquid, and having an inner diameter that tapers on progressingdownwardly to a defined-volume tube end region having a substantiallyuniform diameter of less than about 500 μm, an elongate fiber having adiameter substantially the same as that of said tube end region, saidfiber being disposed within said tube for axial movement therein betweenraised and lowered positions at which the fiber's free end is disposedabove and below said tube end region, respectively, and an actuatormovable between two positions, said actuator operatively connected tosaid fiber for shifting the same between its raised and loweredpositions, whereby a defined volume of liquid contained in said tube endregion is expelled from the tube onto such a substrate disposed belowthe tube.
 35. Valving apparatus for use in metering a selected amount ofliquid onto the surface of a substrate, comprising a reservoir forholding such liquid, a tube extending from the reservoir and terminatingat a lower end orifice adjacent a plane adapted to be occupied by thesurface of such a substrate, a fiber disposed in said tube for axialoscillatory movement therein, with a lower portion of the fiberextending through the orifice, and the inner diameter of said tube andthe diameter of said fiber being dimensioned to prevent fluid flowthrough said orifice in the absence of fiber oscillation, and anoscillating unit operatively connected to said fiber for oscillating thesame, including a control unit for determining the oscillationamplitude, frequency and time applied to said fiber, and thereby theamount of liquid allowed to pass through the tube orifice.